Suspension cell lines derived from CHO and HEK 293 cells are commonly used for mammalian protein production. These cell lines have many desirable traits including high expression levels, scalability (density and volume), and especially in the case of CHO cells, a history of regulatory approval.

Clinical biotherapeutics are frequently generated using stable transfectants for batch-to-batch consistency and low cost at extremely large scale. Many advances spanning the last decade such as improved cell lines, expression vectors, culture medium, and delivery methods have led to the adoption of transient transfection methods for mammalian protein expression.

In many drug discovery applications, it is beneficial to screen protein constructs quickly using transient transfection methods, allowing for the evaluation of various target molecules or protein isoforms simultaneously. In many instances, transient transfections are performed in parallel while more resource-intensive stable cell lines are under development.

Recent advances in transfection technologies, which include the TransIT-PRO® Transfection Kit byMirus Bio, have allowed researchers to obtain high protein titers in suspension CHO and 293 derived cells in a simple and reproducible manner. Using Mirus Bio's technology, transfection complexes are prepared in serum-free media by adding plasmid DNA, TransIT-PRO Transfection Reagent, and PRO Boost Reagent.

The PRO Boost Reagent is an optional component and enhances gene expression in certain growth media formulations. After incubating complexes for 10–30 minutes, they can be added directly to cells in normal growth media. Transfection using the TransIT-PRO Transfection Kit eliminates the need for a culture medium change post-transfection and is suitable for both transient and stable transfection. For secreted antibody constructs, optimal titers are typically obtained 5–7 days post-transfection in batch fermentation.

Several parameters should be considered during the optimization of transient transfection protocols including cell density at the time of transfection, DNA concentration, reagent-to-DNA ratio, and cell culture medium.

Cell Density

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Figure 1. Optimal cell density at the time of transfection is 0.5 x 106–1.0 x 106 cells/mL. CHO-S cells were transfected using the TransIT-PRO Transfection Kit at a range of cell densities at the time of transfection. Complexes were formed using a 1:1:1 ratio of TransIT-PRO:Boost Reagent:DNA using an enhanced green fluorescence protein (EGFP) reporter plasmid. GFP transfection efficiency was determined at 48 hours post-transfection using flow cytometry. Transfections were performed in 24-well deep-well shaker blocks using FreeStyle™ CHO-S cells cultured in FreeStyle CHO Expression media (2 mL/well). Error bars represent the standard deviation of triplicate wells.

Cellular health is an important consideration prior to any tissue culture experiment. Ideally, cells should have a consistent doubling time and high viability. Contamination with bacteria, yeast, virus, or mycoplasma will not only impair cellular health but also transfection efficiencies.

For suspension cells that can grow to very high densities, it is important to optimize the cell density at the time of transfection. Efficiencies are highest when cells are actively dividing during transfection, which assists in the nuclear entry of the plasmid DNA. Figure 1 shows a titration of CHO suspension cell densities ranging from 0.25 x 106–2.0 x 106 cells/mL. The maximal number of GFP-expressing cells (approximately 60%) is detected at cell densities between 0.5 x 106–1.0 x 106 cells/mL at the time of transfection.

The plasmid DNA concentration needs to be sufficient to obtain high gene expression. However, extremely high DNA levels can be toxic and/or cost-prohibitive. Figure 2 examines a range of plasmid DNA concentrations from 0.25–3.0 µg per mL of culture using three different doses of TransIT-PRO and PRO Boost Reagent. Typically, 1.0–1.5 µg DNA per mL of culture is the optimal concentration for maximal expression. In some cell types, increased plasmid DNA concentration results in improved transfection efficiencies, therefore the benefit of increased expression needs to be weighed against the added cost.